This invention relates to agents which extend the proliferative capacity of somatic cells and reverse senescent gene expression.
The in vitro proliferative capacity of normal somatic cells is finite. This cessation of proliferation after 60-100 doublings in vitro is reproducible and is a frequently used model of aging on a cellular level (see, e.g., Hayflick and Moorehead, 25 EXP. Cell Res. 585, 1961; Hayflick, ibid., 37:614, 1985; West et al., 184 Exp. Cell Res. 138, 1989). The specific maximum proliferative capacity of a given cell strain is dependent upon the tissue of origin and is inversely proportional to the in vivo age of the donor (see, e.g., Martin et al., 23 Lab. Invest. 86, 1979; Goldstein et al., 64 Proc. Natl. Acad. Sci. USA 155, 1969; Schneider and Mitsui, ibid., 73:3584, 1976; and Le Guilty et al., 19 Gerontologia 303, 1973).
Various theories have been proposed to explain this cessation of proliferation including the free radical theory which suggests that free radical-mediated damage to DNA and other macromolecules is causative in critical loss of cell function (Harmon, 11 J. Gerontol. 298, 1956; Harmon, 16 J. Gerontol. 247, 1961), somatic mutation theories which propose that without genetic recombination cells lack the ability to proliferate indefinitely due to a progressive loss of genetic information (Burnet, "Intrinsic Mutagenesis--A Genetic Approach to Aging", Wile, N.Y., 1976; Hayflick, 27 Exp. Gerontol. 363, 1992), and genetically programmed theories of senescence which suggest that the expression of senescent-specific genes actively inhibit cell proliferation perhaps under the direction of a mitotic clock (Martin et al., 74 Am. J. Pathol. 137, 1974; Goldstein, 249 Science 1129, 1990).
Consistent with the programmed theory, some genes such as collagenase, plasminogen activator inhibitor 1 (PAI-1) and .beta.-amyloid precursor protein (.beta.APP) have been demonstrated to be expressed at a higher level in senescent cells (West et al., Supra; West et al., 31 Gerontologist 313, 1991; West et al., Id. at 355). These gene products in turn are thought to play important roles in age related diseases.
Finlay and Cristofalo, 168 Exp. Cell Res. 191, 1987, and Finlay et al., 156 Exp. Cell Res. 462, 1985, describe a secondary effector molecule produced by cells exposed to glucocorticoid hormones. They indicate that chronic exposure to hydrocortisone or dexamethasone results in a 20-40% extension in the proliferative lifespan of W138 human fetal lung fibroblast cells.
Normally, W138 cells possess a limited replicative lifespan. The authors, Finlay and Cristofalo, indicate that the decline in proliferative capacity of cultures of these cells with serial subcultivation is characterized by (a) a decreased rate of growth, (b) an exponential decrease in a number of cells capable of incorporating tritiated thymidine into DNA, and (c) a decreased saturation density of the culture. The authors further indicate that addition of glucocorticoid hormone to these cells at subcultivation results in both an increase in tritiated thymidine incorporation into DNA during logarithmic growth, and a 20-40% increase in saturation density. No response was observed when glucocorticoid hormone was added during logarithmic growth instead of at subcultivation.
The stimulatory effects of glucocorticoid hormone are said to be mediated by a factor present in the medium conditioned by the cells for 24 hours following subcultivation in the presence of the hormone. This factor is said to be stable at 75.degree. C. for 90 minutes, dialyzable using a 12,000 daltons molecular weight (MW) cut-off tubing, but retained when dialyzed against 3,500 daltons MW cut-off tubing. Increased time of dialysis using the 3,500 daltons MW tubing, however, did result in significant loss of activity. The authors state, "It is possible, therefore, that the factor(s) is of MW less than 3 500." The authors also indicate that the material is resistant to treatment with trypsin, chymotrypsin and protease, and is relatively polar. Finally, the authors indicate, "[I]t appears that peptide components are not present in the stimulatory refractor. If the glucocorticoid-induced factor is a small peptide or glycopeptide, then the peptide components necessary for the stimulatory activity are either not available for proteolytic attack or are not cleaved by the endopeptidases used in this study." (168 Exp. Cell Res. 191, 1987)